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  • ISSN 1006-3080
  • CN 31-1691/TQ

猪流行性腹泻病毒串联表位亚单位疫苗的免疫原性

杨灿灿 吴诗璟 张峒 张元兴 刘琴

杨灿灿, 吴诗璟, 张峒, 张元兴, 刘琴. 猪流行性腹泻病毒串联表位亚单位疫苗的免疫原性[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20210515006
引用本文: 杨灿灿, 吴诗璟, 张峒, 张元兴, 刘琴. 猪流行性腹泻病毒串联表位亚单位疫苗的免疫原性[J]. 华东理工大学学报(自然科学版). doi: 10.14135/j.cnki.1006-3080.20210515006
YANG Cancan, WU Shijing, ZHANG Tong, ZHANG Yuanxing, LIU Qin. Immunogenicity of Porcine Epidemic Diarrhea Virus Tandem Epitope Subunit Vaccine[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20210515006
Citation: YANG Cancan, WU Shijing, ZHANG Tong, ZHANG Yuanxing, LIU Qin. Immunogenicity of Porcine Epidemic Diarrhea Virus Tandem Epitope Subunit Vaccine[J]. Journal of East China University of Science and Technology. doi: 10.14135/j.cnki.1006-3080.20210515006

猪流行性腹泻病毒串联表位亚单位疫苗的免疫原性

doi: 10.14135/j.cnki.1006-3080.20210515006
基金项目: 中央高校基本科研业务费专项(222201211729)
详细信息
    作者简介:

    杨灿灿(1995—),女,河南人,硕士生,研究方向为动物疫苗。E-mail:naccyang@163.com

    通讯作者:

    刘琴,E-mail:qinliu@ecust.edu.cn

  • 中图分类号: Q819

Immunogenicity of Porcine Epidemic Diarrhea Virus Tandem Epitope Subunit Vaccine

  • 摘要: 将纤突蛋白S的COE区域 (E1)、S1D区域 (E2)、C末端区域 (E3) 以及膜蛋白M的M3区域 (E4) 设计成串联表位亚单位 (EC),以报道的COE和S1候选亚单位作为对照,构建了不同亚单位疫苗的杆状病毒表达体系。杆状病毒载体表达系统生产的目标蛋白,采用镍柱亲和层析进行纯化。在BALB/c小鼠上,不同亚单位疫苗的免疫原性初步评价表明,EC、COE和S1序列分别成功插入杆状病毒基因组,EC、COE和S1蛋白均能在Sf9细胞中分泌表达,但是纯化条件依蛋白而有差异。与COE和S1亚单位疫苗相比,EC亚单位疫苗能激发小鼠产生更多的特异性免疫球蛋白IgG、干扰素-γ和肿瘤坏死因子-α。上述结果表明,各个亚单位疫苗均能激发小鼠的体液免疫与细胞免疫,与COE和S1亚单位疫苗相比,EC亚单位疫苗能更好的激发小鼠的体液免疫与细胞免疫。

     

  • 图  1  携带EC、COE或S1序列的重组杆状病毒菌落验证

    Figure  1.  Validation of recombinant baculovirus colonies born with EC, COE or S1 sequences.

    图  2  Western blot 检测COE (a)、EC (b) 和S1 (c) 蛋白在昆虫细胞系Sf9中表达

    Figure  2.  The expressions of COE (a), EC (b) and S1 (c) proteins in insect cell line Sf9 detected by Western blot.

    Western blot detected the expression of COE, EC and S1 proteins in the insect cell line Sf9. Each protein had a 6× His tag, and His antibody was used to detect the target protein. 1: The culture supernatant of sf9 cells infected with recombinant baculovirus; 2: The lysate supernatant of sf9 cells infected with recombinant baculovirus; 3: The lysate precipitation of sf9 cells infected with recombinant baculovirus.

    图  3  SDS-PAGE检测镍柱亲和层析纯化的EC (a)、COE (b) 和S1 (c) 蛋白

    Figure  3.  SDS-PAGE determinations of EC (a), COE (b) and S1 (c) proteins purified with Ni affinity chromatography column.

    Y−Pre-column sample; L−Flow-through sample; 10, 15, 50, 100, 500: 10, 15, 50, 100, 500 mmol/L imidazole eluent, respectively.

    图  4  ELISA检测两次免疫EC、COE或S1亚单位疫苗小鼠血清IgG (a)、IFN-γ (b) 和TNF-α (c) 水平

    Figure  4.  ELISA determination of serum IgG (a), IFN-γ (b) and TNF-α (c) levels of mouse vaccinated with EC, COE or S1 subunit vaccine twice.

    The purified EC, COE, and S1 proteins were mixed with aluminum gel adjuvant in equal volume to prepare subunit vaccines. The vaccines of each group and the PBS group were subcutaneously injected into BALB/c mouse. The first immunization dose was 75 µg, the second immunization was performed two weeks after the first immunization, and the second immunization dose was 50 µg. ELISA detected serum IgG, IFN-γ and TNF-α levels of immunized mouse to evaluate the immunogenicity of different subunit vaccines.

    表  1  引物信息

    Table  1.   Primers information

    Primer namesPrimer sequences (5’→3’)
    signal-FGGCGCGGGATCCATGCGCTCACTGATCTAC
    signal-RGCAGAGTCACACGGGTCACGTCCTGAGGCA
    E1-FCGTGACCCGTGTGACTCTGCCCTCATTCAA
    E1-RCATGAAGGAGACGTCAGTGCTACCGCCTCCACCGACGTCAGTGAT
    E1-R1GTGATGGTGATGGTGATGGACGTCAGTGATGCCTTCCAGAGGC
    E2-FTAGCACTGACGTCTCCTTCATGACCCTGGA
    E2-RGAAAGCCTCGTAGGGCTGCAGGCTACCGCCTCCACCGATAGACATGCT
    E4-FGTAGCCTGCAGCCCTACGAGGCTTTCGGTGGAGGCGGTAGCGTGCAGGTC
    E4-RGTGATGGTGATGGTGATGCACCAGGTGCAGGACCTTCTCGGAGTCAGT
    S1-RGTGATGGTGATGGTGATGGTGGTAGAAGAAACCTGGCAGTTCA
    P-EC-FGTGCATCACCATCACCATCACTAAAAGCTTGTCGAGAAGTACTAGAG
    P-E1-FGTCCATCACCATCACCATCACTAAAAGCTTGTCGAGAAGTACTAGAGGA
    P-S1-FACCATCACCATCACCATCACTAAAAGCTTGTCGAGAAGTACTAGAG
    P-EC-RTCAGTGAGCGCATGGATCCCGCGCCCGATGGTGGGACG
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出版历程
  • 收稿日期:  2021-05-15
  • 网络出版日期:  2021-07-09

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